Authors

J Nalepa-GrajcarJ Pachebat²; S Hadfield³; E Guy³; M Swain²;  ¹ Aberystwyth University - IBERS, UK;  ² Aberystwyth Uinversity, UK;  ³ Public Health Wales, UK

Discussion

Toxoplasma gondii (T. gondii.) is protozoan parasite which infects approximately one third of the world’s population. Infections usually result in mild symptoms or are asymptomatic, but can be severe or life threatening in clinically vulnerable groups. A cost effective method of performing whole genome sequencing (WGS) directly from clinical samples is highly desirable. WGS of T. gondii has been successful using organisms cultured in laboratory animals. In clinical samples, human DNA is at relatively high levels as compared to T. gondii DNA, which is at relatively low levels. The ratio of human to T. gondii DNA is unfavourable – WGS of these samples generates lots of human sequences, and very few Toxoplasma sequences. This approach is too expensive and wasteful to be widely used.  There is, however, a relatively new method known as Selective Whole Genome Application (SWGA) that can overcome these problems. It involves designing primers that preferentially amplify the parasite DNA (i.e.  T. gondii) over that of the contaminating host DNA (i.e. human). It has been successfully employed to amplify Plasmodium spp. directly from clinical blood samples (Cowell et al. 2017). 

We attempted to apply the SWGA method to T. gondii samples cultured in vitro in human HFF cells and primary results looks very promising. We have already designed primer sets to amplify SWGA to target T. gondii. DNA from human host DNA. We have identified primers that can be combined into primer sets and have performed the amplification and WGS, allowing characterization of isolates that would otherwise have been expensive or impossible to sequence.

We achieved high an average genome coverage, with up to 95% of the T. gondii core genome. Preliminary results show that SWGA with designed primer sets results in a preferential increase in the amount of target Toxoplasma DNA compared to the background human DNA. 

Demonstrating via WGS that the method works with clinical samples would be a significant breakthrough for the field. It would allow us to perform WGS on an extensive library of clinical samples possessed by Toxoplasma Reference Unit (TRU) Public Health Wales. These resources would enable significantly more in-depth investigations, improving understanding of the epidemiology, virulence and other traits of this important human pathogen that has not otherwise been possible. 

We believe we are the first group to apply SWGA to T. gondii. By applying our SWGA method to the clinical samples from the TRU, we will demonstrate its wide applicability to critical medical needs.